This invention relates to a method for determining HIV susceptibility to protease inhibitors.
Treatment of the Human Immunodeficiency Virus type 1 (HIV-1) infection by antiretrovirals can lead to the selection of virus variants with decreased susceptibility to these agents. Near complete inhibition of HIV replication in vivo by triple combinations of reverse transcriptase inhibitors (RTIs) and of protease inhibitors (PIs), now often termed highly active antiretroviral therapy (HAART), is believed to significantly prevent emergence of resistance. However, in many patients treated by such drug combinations, HIV resistance to both RTIs and PIs can gradually develop, which appears to be mostly the case for patients in which the antiviral combination has failed to achieve a complete block of viral replication. In such patients, optimally active alternative treatment regimens need to be found, which will require careful measurement of HIV susceptibility to antiretroviral drugs. In addition, since antiviral combination therapy needs to be optimized to ensure the best long-term antiviral response, it could be essential to monitor HIV susceptibility profiles to antiretrovirals before the onset of therapy.
The monitoring of HIV resistance to antiretrovirals can be performed by genotypic analysis of the protease (PR) and of the reverse transcriptase (RT) coding regions of viral genomes carried in the plasma of infected patients. Nevertheless, the number of mutations in PR or in RT that are able to affect HIV susceptibility to a growing number of antiviral molecules is increasing considerably. Hence, the observed combinations of mutations, which are certainly an important marker of the evolution of resistance in the course of the treatment, cannot give precise indications on the actual level of susceptibility or resistance of the studied virus. Instead, only phenotypic assays, which directly measure inhibition of virus replication by antivirals in culture, can provide a quantitative assessment of resistance.
Several of the currently used phenotypic resistance assay systems examine the susceptibility of virus isolates obtained by coculture of patient blood cells with donor primary peripheral blood mononuclear cells (PBMCs). These methods require several rounds of virus growth in primary donor cells for virus amplification, titration, and subsequent testing in the presence of the drugs. As a consequence they are costly and highly time-consuming.
More recently, an innovative technique, the recombinant virus assay (RVA), based on previous observations showing that deletions in transfected retroviral genomes could be repaired by homologous recombination, was proposed by Kellam and Larder. In this system, PCR-amplified viral RT sequences from patient plasma were cotransfected with a RT-deleted infectious molecular clone of HIV yielding a recombinant virus carrying the RT sequences of patient plasma virus. Since the molecular clone used for recombination was initially obtained from a laboratory-adapted HIV-1 strain, the recombinant virus could be conveniently tested on established cell lines instead of primary cell cultures.
Several versions of the RVA have been developed for HIV-1 susceptibility testing, which have yielded EC50 values that correlate well with corresponding HIV-1 RT or PR genotypes. However, in these recombinant systems, as well as in the PBMC assay, production of a testable stock of infectious particles requires amplification and titration of virus produced by exponential growth in lymphocytic cells, a step that requires cumulative rounds of viral replication and promotes genetic drift of the virus. Since several mutations able to confer resistance to RTIs or PIs have been shown to reduce the replicative capacity of the virus and to be selected against during drug-free HIV replication, there exists a need in the art for a susceptibility assay for HIV that would only require one step of virus replication.